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WOUND HEALING
2012
CONCEPTS
Wound healing is a complex process that normally occurs in the
postnatal setting through scar tissue formation, with regenerative healing limited
to the liver and bone.
In contrast, the fetus in the mild-gestational period heals cutaneous
wounds without scarring by regeneration of the normal dermal architecture,
including restoration of dermal appendages and neurovasculature, in all
mammalian species. This period of regenerative healing is followed by a
transitional period in which wounds heal with a normal extracellular matrix but
fail to regenerate its dermal appendages. Lastly, near the end of gestation,
progression to the postnatal phenotype exists in which wounds heal with an
excess of collagen, a loss of dermal appendages and a flattened epidermis.
Many studies have shown that stimulating inflammation enhances the
extent of scarring in fetal wounds. Moreover, a more substantial inflammatory
response to injury is seen in late-gestational fetal skin that heals with a scar.
These findings suggests an intrinsic property of fetal skin that is permissive of
scarless wound healing.
Limb regeneration is one of the best examples of organ/appendage
regeneration in vertebrates and has been called “epimorphosis” or “epimorphic
regeneration”since it requires blastema formation and proliferation. Urodele
amphilibians, such as newts and salamanders, can regenerate amputate
appendages (limbs and tail). Typically, wound healing proceeds incredibly
quickly in these animals. Within 4 to 12 hours, the stump is covered with a layer
called the “wound epidermis”.
Unlike urodele amphibians, the regenerative ability of anuran amphibians
(frogs and toads) depends on their developmental stage. For instance, before
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metamorphosis can completely regenerate its developing hindlimb buds, this
regenerative capacity declines as metamorphosis proceed.
The skin of mammalian adults can neither heal scarlessly nor regenerate
skin derivates, such as sweat glands or hair follicles. Wound healing in
postnatal mammal's skin (organ) could be considered an unusual regeneration
process since regeneration of the epithelium (parenchyme) is incomplete while
an excessive production of connective tissue (stroma) is associated. Therefore,
skin wound repair is defective, since a tissue forms lacking the structural and
functional characteristics of normal skin. Scar formation, unfortunately presents
the main form of repair in adult skin wound healing.
Every tissue disruption of normal anatomic structure with consecutive
loss of function can be described as a wound. Tegmental injuries are defined as
open or outer wounds, whereas inner or closed wounds are injuries or ruptures
of inner organs and tissues with the skin still intact.
It is well known that the difference between superficial and deep wounds
is of great clinical importance and largely determines how these injures heal
and the degree of scarring to be expected. Superficial wounds usually heal with
a minimum of scarring. Superficial injury less than 0.56 mm in depth or 33% of
normal hip skin thickness, results in regeneration rather than scar, whereas
deeper injury results in increasing scar formation. This result suggests that
injury beyond a critical depth leads to scar formation rather than regeneration.
The stages of wound repair
The multiple pathophysiological mechanisms that overlap during the
progression of the skin wound-healing reaction may explain the lack of
consensus on the number of phases involved in this reaction. Some researches
argue that wound healing involves three phases: Inflammation, proliferation and
tissue remodeling whereas other researchers believe there are four stages in
wound healing: Hemostasis, inflammation, proliferation and tissue remodeling
with scar formation; and others even defend that there are five phases:
Hemostasis, inflammation, cellular migration and proliferation, protein synthesis
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and wound contraction and remodeling. However, everyone agrees that these
phases are interrelated, suggesting that the wound-healing process is a
continuum.
In the 3-stage model of wound healing, inflammation includes
coagulation and inflammatory cell recruitment. The different clotting cascades
are then initiated by clotting factors from the injured skin (extrinsic system).
Then, thrombocytes get activated for aggregation by exposed collagen (intrinsic
system). At the same time, the injured vessels follow a 5 to 10 minute
vasoconstriction, triggered by the platelets, to reduce blood loss and fill the
tissue gap with a blood clot comprised of cytokines and growth factors.
Furthermore, the blood clot contains fibrin molecules, fibronectin, vitronectin
and thrombospondins, forming the provisional matrix as a scaffold structure for
the migration of leukocytes, keratinocytes, fibroblasts and endothelial cells and
also acts as a reservoir of growth factors. The life-saving vasoconstriction with
clot formation accounts for a local perfusion failure with a consecutive lack of
oxygen, increased glycolysis and pH-changes. The vasoconstriction is then
followed by a vasodilation in which the traumatized tissue suffers a reperfusion
phenomenon. Both platelets and leukocytes release cytokines, chemokines and
growth factors to activate the inflammatory process, stimulate the collagen
synthesis, activate the transformation of fibroblasts to myofibroblasts, start
angiogenesis and support the reepithelialization process. The vasodilatation
can also be recognized by a local redness (hyperemia) and by wound edema
(tumor).
Neutrophil recruitment is crucial within the first days after injury because
their ability in protease secretion and phagocytosis kills local bacteria and helps
to degrade necrotic tissue. They start their debridement by releasing highly
active antimicrobial substances i.e. cationic peptides and eicosanoids, and
proteinases, i.e. elastase, cathepsin G, proteinase 3 and an urolinase-type
plasminogen activator. Approximately 3 days after injury, macrophages enter
the zone of injury and support the ongoing process by performing phagocytosis
of pathogens and cell debris and by secreting growth factors, chemokines and
cytokines. Macrophages have many functions including host defense, the
promotion and resolution of inflammation, the removal of apoptotic cells and the
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support of cell proliferation and tissue restoration following injury. Beside their
immunological functions as antigen-presenting cells and phagocytes during
wound repair, macrophages supposedly play an integral role in a successful
healing response through the synthesis of numerous potent growth factors,
such as transforming growth factor (TGF-, TGF-, basic FGF, platelet derived
growth factor (PDGF) and vascular endothelial growth factor (VEGF), which
promote cell proliferation.
During the proliferative phase, granulation tissue deposition provides a
wound bed for re-epithelialization. In the phase of proliferation, that is 3 to 10
days after wounding, the main focus of the healing process lies in covering the
wound surface, the formation of granulation tissue and restoring the vascular
network. The proliferative phase in wound healing is characterized by
angiogenesis, granulation tissue formation, epithelialization and wound
contraction. Granulated tissue basically consists in fibroblast and new blood
vessels. The first step in new vessel formation is the binding of growth factors to
their receptors on the endothelial cells of existing vessels, thereby activating
intracellular signaling cascades. The endothelial cells proliferate and migrate.
This is a process also known as “sprouting”. The newly built sprouts form small
tubular canals that interconnect with others forming a vessel loop. Thereafter,
the new vessels differentiate and mature by stabilizing their vessel wall via the
recruitment of pericytes and smooth muscle cells.
Towards the end of this stage, fibroblasts, attracted from the edge of the
wound or from the bone marrow, are stimulated by macrophages, and some
differentiate into myofibroblasts. Fibroblasts begin synthesizing collagen and
proliferate to form granulation tissue. TGF- induces fibroblasts to synthesize
type I collagen and reduce matrix metaloproteinases (MMPs) production.
Fibroblasts produce collagen but also extracellular matrix substances, like
fibronectin,
glycosaminoglycans,
proteoglycans
and
hyaluronic
acid.
Myofibroblasts are contractile cells that, over time, bring the edges of a wound
together. Wound contraction is a biological means whereas the edges of an
open wound are pulled together by forces resulting from the wound-healing
process. The environmental cytokines have a significant effect on the contractile
process. TGF- increases the rate and degree of contraction without
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upregulating proliferation and it has been postulated that this may be through
the induction of PDGF. At the end of this phase, the number of fibroblasts is
reduced by myofibroblast differentiation and terminated by consecutive
apoptosis.
Remodeling is the last phase of wound healing and occurs from day 21
to up 1 year after injury. During the remodeling phase, formation of granulation
tissue ceases through apoptosis of the responsible cells. This process is
important because its aberration leads to hypertrophic scarring and keloids. A
mature wound is therefore characterized as avascular and acellular. With
wound maturation, the composition of the extracellular matrix undergoes
change. The type III collagen deposited during the proliferative phase is slowly
degraded and replaced with stronger type I collagen. This type of collagen is
oriented in small parallel bundles and is, therefore, different from the basketweave collagen in healthy dermis.
Later on, myofibroblasts cause wound contractions by their multiple
attachments to collagen while they help decrease the surface of the developing
scar. The angiogenic processes also diminish; the wound blood flow declines
and the acute wound metabolic activity slows down and finally stops. Scar
contraction is the shrinkage that occurs in an already healed scar. Well-known
clinical risk factors include hypertrophic scarring and the most predominant
theories involve myofibroblasts. The results using fibroblast and myofibroblast
isolated from hypertrophic scars, suggests that fibroblasts are the primary cells
involved in wound contracture whereas myofibroblasts are the primary cells
involved in scar contracture.
Connective tissue growth factor (CTGF) is a downstream regulator of
fibrosis that is induced by TGF. It seems, that although TGF is important in
initiating pathologic scarring, it is CTGF that sustains the fibrotic process. Insulin
like growth factor (IGF-1) modulates growth hormone effects on fibroblasts.
IGF-1 is expressed locally in injured tissue formation up to 5 weeks post-injury.
In addition IGF-1 has been shown to act as a TGF- stimulating factor and both
play an important role in the pathogenesis of abnormal scarring.
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Inflammatory phenotypes related to wound repair
In a broader perspective, the inflammatory response would include all
phases that constitute wound healing. We have therefore proposed that
inflammation could be the basic mechanism that drives the nature of the
different stages of wound repair. In essence, the post-traumatic local acute
inflammatory response is described as a succession of three functional phases
of possible trophic significance: nervous or immediate (ischemia-reperfusion
phenotype), immune or intermediate (leukocytic phenotype) and endocrine or
late (angiogenic phenotype) (Figure 1). Thus, one could also hypothesize that
the sequence of transformations undergone by traumatized tissue in the three
above-mentioned phases represents a metamorphic phenomenon. The
inflammatory response induced in the wounded skin could be a result of three
overlapping phases during which metabolic phenotypes featuring progressive
complexity of oxygen use are expressed.
Figure 1: The Inflammatory Response.
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1. The ischemia-reperfusion phenotype
In the first or immediate phase is referred to as the nervous phase
because the sensory (pain and analgesia) and motor alterations (contraction
and relaxation) respond to the injury. Wounds produce a pathological
neuromuscular response that induces systemic and local ischemia-reperfusion
through sensory changes (stress, inflammatory pain, analgesia) and motor
alterations with skeletal muscle reactions (fight-to-flight and withdrawal
reflexes); myocardium changes (tachycardia) and vascular smooth muscle
impairment (vasoconstriction and vasodilation). Sudden hydroelectrolytic
change is a common and basic pathogenic mechanism of this response.
Through this mechanism, the intense local and systemic inflammatory
responses are associated with abnormal ion transport.
In the early neurogenic stress response, the activation of the
hypothalamic-pituitary-adrenocortical, sympathetic-adrenal medullary and reninangiotensin-aldosterone axes occur, with the release of catecholamines,
glucocorticoids and mineralocorticoids. Consequently, selective accumulation of
these substances in the interstitial space of tissues suffering from ischemiareperfusion is produced because endothelial permeability is increased,
especially in postcapillary venules (Figure 2).
Damage inducible alarm signals from injured tissue, such as those
exposed to mechanical damage, may determine the subsequent activation of
this pathological neuro-motor response. Tissue injury is a critical initiator of the
acute inflammatory response. Cell components released by necrotic cells elicit
local, regional and systemic inflammatory responses. Accordingly, the
inflammatory activity of dying cells decays over time.As soon as these alarm or
“danger” signals cannot be replenished, they are degraded. Endogenous
inducers of inflammation include ATP, K+ ions, the nuclear protein HMGB1
(high-mobility group box 1 protein), heat shock proteins, the end product of
purine catabolism, uric acid, galectins and several members of the S100
calcium-binding protein.
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Figure 2:
An early pathological motor response, where the smooth muscular fibers
is prominent, particularly in the vascular system, is triggered. The vasomotor
response with vasoconstriction, which collaborates in the production of ischemia
and vasodilation, causes the redistribution of the local vascular and systemic
blood flow. The intensity and duration of this ischemia-reperfusion phenomenon
determines the evolution of the subsequent inflammatory response phases.
Vasoconstriction occurs in this vasomotor response and produces
ischemia and cellular edema. This is then followed by vasodilation with
reperfusion injury, which in turn causes exudation, followed by an increase in
endothelial permeability that induces interstitial edema. Both cellular, by
ischemia, as well as interstitial, by reperfusion, edema could represent an
ancestral mechanism to feed cells by diffusion.
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Nevertheless, disturbance of ion transport has been associated with
cellular dysfunction. There is increasing evidence that conditions characterized
by an intense local inflammatory response are associated with abnormal ion
transport. Inflammatory mediators which influence ion transport are bradykinin,
leukotrienes, cytokines and TGF. They trigger the release of specific
messengers like prostaglandins, nitric oxide and histamine, which alter ion
transport function through specific receptors, intracellular second messengers
and protein kinases. There is an additional influence of thrombin, and the
complement system.
Interstitial edema causes a steady separation of the cells from the
capillaries, which would favor the persistence of an ischemic phenotype
(anoxia) and the defective use of oxygen (hypoxia) represented by excess
production of reactive oxygen and nitrogen species or oxidative and nitrosative
stress during reperfusion. Oxidative and nitrosative tissue damage could also
increase lipid peroxidation with increased membrane permeability, increased
degradation of extracellular matrix and edema. The accumulation of
glycosaminoglycans fragments has been proposed as an important mechanism
for edema formation because of its hydrophilic properties. Glycosaminoglycans
are long unbranched polyssacharide that tend to adopt highly extended random
coil conformation and occupy a huge volume for their mass. They attract and
entrap water and ions, thereby forming hydrated gels, while permitting the flow
of cellular nutrients. Under inflammatory conditions, hyaluronan, a nonsulphated
glycosaminoglycan, is more polydisperse with a preponderance of lowermolecular forms; it favors edematous infiltration of the tissues as well as the
interstitial fluid flow and the tissue lymph pressure gradient. Likewise, while the
progression of interstitial edema reduces the blood capillary function, it
simultaneously enhances lymphatic circulation (circulatory switch). Also,
interstitial flow is important for lymphangiogenesis. The interstitial fluid flow
associated with edema, even though it can be extremely slow, can have
important effects on tissue morphogenesis and function, cell migration and
differentiation and matrix remodeling, among other processes. Abnormally
increased interstitial flow rates can occur during inflammation and can also
trigger fibroblasts to differentiate or remodel the extracellular matrix, contributing
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to the development of tissue fibrosis. Also, upon activation, mast cells, major
effector cells in host defense responses and immunity, not only release
vasoactive substances, i.e. histamine and serotonin, but also proteolytic
enzymes favoring interstitial edema. Curiously, the functional impotence of the
somatic motor system, which controls voluntary movements, favors vascular
blood stasis and interstitial edema. Limiting swelling is extremely important,
because the injured area cannot return to normal until swelling is gone. In
musculoskeletal injuries, this is best accomplished with the “RICE” technique,
which involves “Rest, Ice, Compression and Elevation”.
The traumatized tissue seems to adopt an ischemic phenotype (anoxichypoxic). Chronic extreme hypoxia leads to tissue loss. In contrast, generally
acute mild to moderate hypoxia supports adaptation and survival. The
traumatized tissue probably suffers a metabolic hypoxia, a state where,
although oxygen is available, the cell is unable to utilize it for respiration. In this
critical situation, HIF- (hypoxia inducible factor) independent mechanisms of
energy conservation, could promote survival under very low oxygen conditions
but they are not compatible with the formation of new tissue, as required during
wound healing. On the contrary, during the subsequent phases of the posttraumatic evolution, HIF-dependent pathways for survival and vascularization
can function under conditions where hypoxia is moderate and not extreme. HIF1 enhances the expression of hypoxia responsive genes and therefore, allows
improved cell survival in conditions of limited oxygen availability. HIF-1
activates the transcription of genes involved in diverse aspects of cellular and
integrative physiology, including energy metabolism, cell growth, survival,
invasion, migration and angiogenesis.
Hence, in this initial phase of the inflammatory response, it could be
considered that hypometabolism, anerobic glycolysis with lactate production,
low temperature and decreased energy expenditure are associated with
primitive cellular trophic mechanisms, which may be favored by neuroendocrine-stress response substances to arrive to the interstitial space of the
traumatized patient. This environment could favor the cell dedifferentiation
process through which cells adapt embryonic characteristics
recruitment with origin in the bone marrow.
and stem cell
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2. The leukocytic phenotype
Nowadays, the inflammatory bone marrow-related response induced by
wounds is considered both a key and complementary arm of the stress
response. The inflammatory activation of the bone marrow stem cell niche
indicates the stimulation of hematopoietic stem cells (HSCs) and mesenchymal
stem cells (MSCs), both multipotent stem cells. HSCs are the progenitors
signaling molecules, including interferons, that appear to stimulate HSC
proliferation in response to acute and chronic inflammation.
The leukocytic phenotype of the acute post-traumatic inflammatory
response is characterized by the infiltration of the traumatized tissue, which has
previously suffered ischemia-reperfusion, by inflammatory cells and bacteria.
Acquiring an active immune phenotype through the traumatized tissue involves
both parenchymal (epithelial cells) and non-parenchymal cells (endothelial cells,
fibroblasts and tissue-resident macrophages, mast cells and lymphocytes) as
well as blood cells that migrate to the tissue interstitium. This interstitial
infiltration occurs in an oxygen-poor environment and one of its purposes could
be trophism of the traumatized tissue (Figure 3).
Cellular interstitial infiltration is favored by the action of intrinsic and
extrinsic components of the coagulation cascades. This results in the production
of thrombin and conversion of fibrinogen of intravascular origin to fibrin. In most
pathophysiological situations, it seems that the activation of both the
coagulation and complement cascades occur simultaneously. The complement
and coagulation systems are organized into proteolytic cascades composed of
serine proteases of the chymotrypsin family. An interesting hypothetical
explanation for the structural and functional similarities between the
complement and clotting systems is that they originate from a common
ancestral developmental-immune cascade. Thus, the functional linkages
between development, immunity and hemostasis in vertebrates would be
explained. Complement activation could express successive and predominant
functions related to the inflammatory post-traumatic phenotypes. Therefore, the
different functions of the multiple components of the complement activation
could be integrated into the acute inflammatory response phases which would
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facilitate the comprehension of the implied biochemical mechanisms, like the
complement-coagulation interactions. Hence, an early provisional would matrix
of fibrin and extravased plasma fibronectin is formed, which also includes other
components, such as the extracellular matrix proteins, vitronectin and
thrombospondin. Blood clots facilitate extravascular migration, first of platelets
and later of leukocytes.
Figure 3:
Within minutes of wounding, inflammatory cells are attracted by
complement activation, degranulation of platelets and products of bacterial
degradation. Neutrophils arrive first, followed by mast cells and monocytes that
subsequently differentiate into tissue macrophages. In the tissue suffering
oxidative stress, symbiosis of the inflammatory cells and bacteria for
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extracellular digestion by enzyme release (fermentation) and by intracellular
digestion
(phagocytosis)
could
be
associated
with
enzymatic
stress.
Furthermore, lymphatic circulation plays a major role in which macrophages,
dendritic cells and mast cells migrate to the lymph nodes and activate
lymphocytes.
Accumulating evidence demonstrates that platelets contribute to the
initiation and propagation of the inflammatory process. These cells are replete
with secretory granules, -granules, dense granules and lysosomes. The granule is the most abundant and its content includes both membrane bound
proteins that become expressed on the platelet surface and soluble proteins
that are released into the extracellular space. Whereas platelet dense granules
contain high concentrations of low molecular weight compounds that potentiate
platelet activation i.e. ADP, serotonin and calcium, -granules concentrate large
polypeptides i.e. fibrinogen and von Willebrand factor, which contributes to
hemostasis. Platelet -granules also influence inflammation both by expressing
receptors that facilitate adhesion of platelets with other vascular cells, i.e. Pselectin, and by releasing a wide range of chemokines, among which CXCL4
and CXCL7 are the most abundant. Perhaps the strongest evidence that
platelet release promotes wound healing is the use of the “platelet-derived
wound healing factor” (PDWHF) in the treatment of chronic wounds. Lastly,
platelet -granules contain a variety of both pro- and anti-angiogenic proteins.
Growth factors stored in -granules include vascular endothelial growth factor
(VEGF), platelet-derived growth factor (PDGE) fibroblast growth factor (FGF),
epidermal growth factor (EGF), hepatocyte growth factor (HGF) and insulin-like
growth factor (IGF).
In the post-traumatic local inflammatory response, the activation of the
innate immune system is not only based on the recognition of danger signals or
danger-associated molecular patterns (DAMPs), but also relies on the presence
of pathogen-associated molecular patterns (PAMPs). DAMPs and PAMPs are
recognized by pattern-recognition receptors (PRRs), that are cytoplasmic,
membrane-bound or secreted. The most intensely studied PRRs are the Tolllike receptors (TLRs), the nucleotide-binding and oligomerization domain
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(NOD)-like receptors (NLRs) and the retinoic acid-inducible gene 1 (RIG-I)-like
receptors (RLRs). In particular, NLRs from central molecular platforms that
organize signaling complexes, such as inflammosome, were coined to describe
the high molecular weight complex that activates inflammatory cascades and
cytokine IL-1B. All these receptors activate signaling cascades that lead to the
activation of MAP kinases and NF-kB. The host can discriminate between
danger and pathogen-associated molecular patterns.
Once activated, TLRs induce different signaling cascades depending on
the adaptor protein, ultimately leading to the activation of the transcription
factors NF-kB, AP-1 and interferon-regulatory factor (IRF). TLRs recognize
PAMPs and DAMPs. Thus TLRs appear to regulate inflammatory responses
during wound healing under both sterile and non-sterile conditions. Ischemiareperfusion injury with oxidative stress, represents the scenario in which a
profound injury-promoting role of TLR-2 and TLR-4 has been most thoroughly
established. Available data suggests that the presence of TLR ligands during
wound healing responses promote healing by scarring rather than healing by
regeneration. The regulatory event of NF-kB activation is the phosphorylation of
IkB proteins by the IkB kinase (Ikk) complex, which leads to IkB protein
ubiquitylation and subsequent degradation. This results in the release of
cytoplasmic NF-kB complexes, which then translocate to the nucleus and drive
the expression of target genes. Thus, the expression of cytokines, cytokine
receptors, chemokine, chemokine receptors, adhesion molecules and autacoids
in the traumatized tissue is induced. Although NF-kB fulfills its expected role in
inducing pro-inflammatory gene-expression programs, surprisingly it also plays
an important role in the resolution of inflammation and tissue repair.
Leukocytes transverse the subendothelial basement membrane during
their immunological surveillance patrol through tissues. This process, called
“diapedesis” is strongly enhanced under the influence of inflammation. The
preferred sites of leukocytes are the venules, which are characterized by a
special composition of the subendothelial basement membrane. Leukocyte
migration into the interstitium occurs in three stages: rolling along the
endothelial surface, firm adhesion and transmigration. In the interstitium, the
recruited and activated neutrophils begin debridement of devitalized tissue and
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attack infectious agents. To perform this task, they release a large variety of
active antimicrobial substances i.e. ROS, cationic peptides, eicosanoids; and
proteases, i.e. elastase, cathepsin G, proteinase 3 (PR-3), urokinase-type
plasminogen activation (uPA). The most probable mechanism by which
neutrophils delay the healing process is through the production of excessive
toxic molecules, like ROS and proteases, which damage the cells of the wound
site and thereby delay the healing process.
Tissue injury is a critical initiator of the acute phase response.
Inflammatory cytokines trigger peutraxin generation. Pentraxins are superfamily
of acute phase reactants. The C-reactive protein (CRP) and serum amyloid Pcomponent are well-characterized short pentraxins. Both resident and innate
immunity cells produce pentraxin 3 in peripheral tissues in response to
inflammatory signals and TLR activation. Neutrophils store pentraxin 3 in
specific granules and release it in response to inflammatory signals. Besides a
protective effect, pentraxins also influence the resolution phases of the
inflammatory response later on.
As monocytes extravasate from the blood vessel they become activated
and differentiate into mature tissue macrophages. The differential activation of
macrophages is involved in many facets of tissue injury and inflammation.
“Classically activated macrophages”, or M1 macrophages, express proinflammatory cytokines (IL-1, IL-6, IL-23, IFN-8) and reactive oxygen/nitrogen
species which are involved in the phagocytosis and killing of microbes. They
also promote type I immune responses. “Alternatively activated macrophages”,
or M2 macrophages, fail to express pro-inflammatory mediators and are
involved in angiogenesis, tissue remodeling and inflammation resolution, and
therefore are supposed to promote repair functions.
T-helper cells play critical roles in modulating the differential activation of
macrophages. Type 1 T-helper (Th1) cells produce pro-inflammatory cytokines
(IFN-, TNF-) which skew macrophages into the M1 phenotype. Type 2 Thelper (Th2) cells belong to a larger spectrum of distinct Th responses that have
evolved to protect the host against a spectrum of pathogens. Traditionally, Th2
cells have been defined as the T cells that produce IL-4, IL-5, IL-13, IL-9 and IL-
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10 and express the transcription factors GATA-binding protein 3 (GATA-3),
signal transducer and activator of transcription-5 (STAT-5) and STAT-6.
However, single-cell analysis has revealed a marked degree of heterogeneity in
the cytokine profiles of Th2 cells. Many endogenous molecules released by
tissue damage are potent inducers of type 2 responses. HMGB-1, a non-histone
chromatin binding protein that is released upon cell death and matrix
metalloproteinase (MMP)-2 activate dendritic cells via a TLR-4 dependent
pathway to induce Th2 cell response. It has been proposed that type 2
responses represent a rapid repair response to tissue damage i.e. synthesis of
collagen I and III, suppression of IL-17 production, enhanced expression of IL10 and generation of anti-inflammatory macrophages, all of which lead to rapid
tissue repair.
Although neutrophils, macrophages and T lymphocytes are considered
central in the pathogenesis of post-traumatic inflammation, recent studies also
imply the involvement of dendritic cells, mast cells, basophils and B
lymphocytes to modulate the inflammatory response and wound healing. In
addition, when leukocytes infiltrate inflamed tissues, they express metabolic
anatomy as well as neuroendocrine ability. Particularly the potential role of
leukocyte-derived neuropeptides and hormones in inflammation as a localized
hypothalamic-pituitary-like axis has been proposed. However, the incorrect use
of oxygen persists in this phase. Activated phagocytes would require anaerobic
glycolisis as the main source of ATP to function, suggesting that activated
leukocytes can metabolically adapt to the hypoxic environment in this evolutive
phase of inflammation.
3. The angiogenic phenotype
Angiogenesis permits numerous substances, including hormones, to be
transported by the blood circulation. For this reason it is considered that the
predominance of angiogenesis during the last phase of the inflammatory
response would allow for naming this period, the “endocrine” phase.
Angiogenesis is defined as the growth of new vessels from preexisting ones
and occurs by sprouting, intussusceptions (non-sprouting) and looping from preexisting vessels. The angiogenic switch which stimulates new vessel formation
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is induced under conditions of hypoxia, low pO2 and mechanical stress i.e.
shear stress. Hypoxia induces angiogenesis through HIF-1 pathway with growth
factor expression elevated in hypoxic conditions in a HIF dependent manner.
Hematopoietic stem cell and endothelial precursor cells (EPC) share a common
stem cell origin from hemangioblasts and are present in bone marrow. During
angiogenesis circulating and pre-existing EPC cells are attracted to the site of
injury and differentiate into endothelial cells.
Although the final objective of endothelial growth is to form new mature
vessels for oxygen, substrates and blood cells, other functions could also be
carried out before the new vessels are formed. Thus, in the initial phases of the
inflammatory response, the new endothelial cells could have antioxidant and
anti-immune properties favoring the resolution as well as the progression of the
angiogenic vascular phenotype. Angiogenesis is regulated by numerous
“classic” factors, including VEGF-A, also known as vascular permeability factor
(VPF), FGF-2, TGF, angioproteins, PDGF, thrombospondin-1 and angiostatin.
Non-classic endogenous stimulators of angiogenesis include erythropoietin,
angiotensin II, endothelins, adrenomedullin, adipokines (leptin, adiponectin),
neuropeptide-Y, vasoactive intestinal peptide (VIP) and substance P. VEGF and
FGF-2 occupy the center stage in the angiogenesis field. They act in synergy to
stimulate endothelial cell function during angiogenesis in tissue repair.
Granulation tissue formation starts about three or four days after injury.
The main cell types driving the generation of the new tissue are macrophages,
endothelial cells, fibroblasts and kertinocytes. Angiogenesis is associated with
granulation tissue formation, as the newly forming cellular complex must be
supplied with oxygen and nutrients. As granulation tissue forms in the healing
wound, the vascular cells intermingle with the provisional matrix, which is
composed mainly of fibrin, fibronectin and vibronectin. Then the new blood
vessels associated with fibroblasts and macrophages replace the fibrin matrix
with granulation tissue, forming a new substrate for keratinocyte migration.
As the inflammatory response regresses by certain stop signals at
appropriate checkpoints, edema production and leukocyte traffic into the
traumatized tissue are prevented. The pro-inflammatory mechanisms probably
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are counterbalanced by endogenous anti-inflammatory signals that serve to
temper the severity and limit the duration of the early phases, which leads to
their resolution, an active rather than a passive process. The resolution of the
inflammatory response is mainly mediated by families of local-activity mediators
that are biosynthesized from essential fatty acids eicosapentaenoic acid and
docosahexaenoic acid. These resolution mediators were termed resolvins and
protectins.
Inflammation
resolution
is
also
mediated
by
lipoxins,
trihydroxystearin-containing eicosanoids that are generated within the vascular
lumen through platelet-leukocyte interactions. Lipoxins were the first mediators
identified to have both direct anti-inflammatory and proresolving properties. The
spectrum of lipoxin’s anti-inflammatory activity, particularly lipoxin A4, include
inhibiting the production of ROS, attenuating the microvascular fluid leak,
attenuating the expression of adhesion molecules on leukocytes and endothelial
cells, diminishing the production of chemokines and preventing neutrophil
adhesion and transmigration through the endothelium and stimulating the
uptake of apoptotic polymorphonuclear leukocytes by macrophages.
It has been also proposed that regulatory T cells (Treg cells) have
evolved to provide a complementary immunological arm to a physiological
tissue-protecting mechanism driven by low oxygen tension (i.e. hypoxia) in
inflamed tissues. The hypoxia-adenosinergic pathways might govern the
production of immunosuppressive molecules that have already been implicated
in the activities of Treg cells. In this way, by virtue of acting in hypoxia and
extracellular adenosine-rich tissue, Treg cells could exert their suppressive
function
with
local
downregulation
of
immune
response,
inducing
“immunodormancy” and protecting tissues from continuing collateral tissue
damage, thus improving healing. Also B-1 lymphocytes, which represent the
main B-lymphocyte population in the peritoneal and pleural cavities, secrete IL10 and exert a downregulatory effect on inflammation and wound healing in
mice.
The progressive resolution of the inflammation favors wound reepithelization. This begins one to two days after injury by the migration of
keratinocytes from the epidermis at the wound edge and from injured
appendages. Keratinocytes at these epidermal fronts move forward between
19
the injured dermis and the fibrin clot. Epithelial cell migration requires the
disassembly of desmosomes and hemidosmosomes, which provide anchorage
of the basal keratinocytes with neighboring epithelial cells and the underlying
basement membrane respectively. This disassembly and keratinocyte migration
requires a cross-talk between growth factors, metalloproteinases, integrins and
structural proteins. In addition to lamellipodia extension, basal keratinocytes
leapfrog over the basal cell near the wound. The keratinocytes that are behind
the leading edge in larger wounds proliferate and mature and finally restore the
barrier function of the epithelium. This could involve the proliferation of
epidermal stem cells.
The migration and proliferation of epithelial cells occurs under the control
of EGF, TGF- and PDGF. Cell migration may also require the upregulation of
tissue
plasminogen
activator,
urokinase
plasminogen
activator
and
metalloproteinases (MMP-1, MMP-9, MMP-10). All these factors act as
molecular scissors and help regulate the provisional matrix, collagen
degradation and cellular movements. Lastly, integrins are also important for
wound re-epithelialization, particularly 1-integrins. It has been proposed that
the phenotypic impairments developed by the keratinocytes during reepithelialization constitute a partial epithelial-mesenchymal transition. Following
the completion of wound-repair, keratinocytes revert their mesenchymal-like
phenotype to the epithelial phenotype.
The interstitium is considered the battle field where inflammation
develops and the stroma is its equivalent in tissues and organs. At the same
time, the most abundant cell type of tissue stroma is the fibroblast, an active
heterogeneous population of cells.
Fibroblasts can also contribute to the resolution of inflammation by
withdrawing survival signals and normalizing chemokine gradients, thereby
allowing infiltrating leukocytes to undergo apoptosis or leave the tissues through
the draining lymphatics. Lastly, fibroblasts may also provide important positional
signals for wound healing and tissue regeneration. In addition to their role in
producing an extracellular matrix, they may facilitate angiogenesis by
production and release growth factors.
20
Fibroblasts, which deposit large amounts of extracellular matrix,
repopulate the wounded area in parallel to angiogenesis. Several days after
injury, a subset of wound fibroblasts differentiate into myofibroblasts which are
responsible for wound contraction and for the deposition of additional matrix
proteins. Transforming growth factor-beta (TGF-) and platelet-derived growth
factor
(PDGF)
initiate
phenotype
changes,
converting
fibroblasts
into
myofibroblasts. Matrix formation requires the removal of granulation tissue with
revascularization. A framework of collagen and elastin fibers replaces the
granulation tissue and leads to progressive tissue sclerosis. This framework is
then saturated with proteoglycans and glycoproteins. This is followed by tissue
remodeling involving the synthesis of new collagen, mediated by TGF-, and
the breakdown of old collagen by PDGF. During the tissue remodeling phase,
the initial collagen type III present in the granulation tissue is gradually
substituted and dominated by collagen type I and the resultant larger collagen
fibrils are abnormally arranged in parallel bundles. Remodeling begins two to
three weeks after injury and lasts for a year or more. Most of the endothelial
cells, macrophages and myofibroblasts, undergo apoptosis, leaving a mass that
contains few cells and consists mostly of collagen and other extracellular-matrix
proteins. However, the prognosis of extensive and deep wounds is not entirely
satisfactory because of scar formation and the loss of normal function and skin
appendages. Therefore, reducing the formation of scars and reestablishing the
normal anatomy and function of the skin and its appendages have become the
aim of regenerative medical research.
During the evolution of the inflammatory response, the traumatized tissue
losses its more specialized functions and structures. In this progressive
deconstruction a redistribution of immediate constituents is produced. In this
context, the redistribution of metabolic resources responds to the different
trophic requirements of the traumatized tissue as the inflammation progresses.
It has been proposed that progressive tissue deconstruction is developed,
among other mechanisms, by autophagy. This process is unique since it
converts topologically intracellular material into topologically extracellular
material. Most of the autophagy genes are present in higher eukaryotes,
indicating that this process has been evolutionarily conserved. However,
21
consumption of the substrate deposits and the dysfunction or failure of the
specialized cells in the traumatized tissues could also represent an accelerated
process of dedifferentiation. The hypothetical ability of the tissues to involute or
dedifferentiate could represent a return to early stages of development.
Therefore it could develop an effective defense mechanism against injury since
it could make retracing a well-known route possible i.e. the prenatal
specialization phase during the evolution of the inflammatory response. This
specialization would require a return of the prominence of oxidative metabolism
with a subsequent angiogenesis in the affected tissue to create the capillary bed
that would make regeneration of the specialized tissue possible.
The ability to use oxygen in the oxidative metabolism is recovered when
the tissues recover their capillary function and, therefore, nutrition is mediated
by them. This type of metabolism is characterized by a large production of ATP
(coupled reaction), which is used to drive multiple specialized cellular processes
with limited heat generation. It would determine the onset of healing.
The blood cells that occupy the interstitial space in this latter phase of the
inflammatory response are red blood cells. To carry out this interstitial
occupation, the red blood cells are transported by the newly formed blood
capillaries and, therefore, angiogenesis is considered to play the main role in
this inflammatory period.
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